Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), a natural product of the Capsicum species of red peppers, is known to induce apoptosis and suppress cell growth in various cancer models. The chemopreventive mechanism of capsaicin is cell specific, and its efficacy is dependent on cell or tissue context. Non-steroidal anti-inflammatory drug (NSAID) activated gene-1 (NAG-1) is a cytokine associated with pro-apoptotic and anti-tumorigenic properties in colorectal cancer. NAG-1 expression is inversely correlated with the occurrence of polyps and tumor, supporting that NAG-1 is a promising molecular target in chemoprevention. Our preliminary data demonstrate that capsaicin leads to suppression of cell growth, up-regulation of NAG-1 and down-regulation of cyclin D1 in human colorectal cancer cells. In specific aim 1, we will investigate the molecular mechanism of capsaicin's anti-tumor effect by characterizing how capsaicin up-regulates NAG-1 and down-regulates cyclin D1 in in vitro studies. We will focus on the roles of PKC, GSK3[unreadable], and C/EBP[unreadable] mediating the stimulatory effects of capsaicin on NAG-1 transactivation. In addition, suppression of cyclin D1 and [unreadable]-catenin signaling by capsaicin will be determined at transcriptional as well as post-translational levels, focusing on [unreadable]-catenin degradation and translocation, or interaction with E-cadherin. We anticipate in vitro studies will provide us a new anti-tumorigenic pathway of pro-apoptotic and anti-proliferative regulation by capsaicin. In our preliminary data, we found that capsaicin had synergistic activity with 3,3'- diindolylmethane (DIM) in NAG-1 activation and suppression of colorectal cancer cell growth. We will investigate the effects of capsaicin alone or in combination with DIM on the development of colorectal cancer using a colorectal tumor model in which [azoxymethane will be treated with capsaicin, DIM, or capsaicin + DIM into wild type or NAG-1 knockout mice] for 4 weeks. Significant reductions in aberrant cryptic foci (ACF) will be expected in mice treated with capsaicin or DIM, and a greater reduction of ACF formation in capsaicin + DIM treated mice is expected than that in mice treated with capsaicin or DIM alone. [Knockout of the NAG-1 gene is expected to ameliorate capsaicin/DIM-induced antitumorigenic activities]. In vivo studies will reveal the additive or synergistic effect of capsaicin and DIM in suppression of colorectal tumorigenesis.